Comparison Evans Blue injection routes: Intravenous versus intraperitoneal, for measurement of blood–brain barrier in a mice hemorrhage model

doi:10.1016/j.jneumeth.2010.12.013

Journal of Neuroscience Methods

Volume 195, Issue 2, 15 February 2011, Pages 206-210

https://doi.org/10.1016/j.jneumeth.2010.12.013 Get rights and content

Abstract

Aims

Intracerebral hemorrhage is one of the most devastating subtypes of stroke, leaving survivors with severe neurological deficits. Disruption of the blood brain barrier (BBB) following hemorrhage results in the development of vasogenic brain edema, a most life-threatening event after such events as intracerebral hemorrhage (ICH). The Evans Blue assay is a popular method for the quantification of BBB disruption. Although this method is in common use, there are several protocols of the assay in the literature which vary in the route of administration, as well as the circulation time of the stain. In this study, we compared the amounts of accumulated stain in brain tissue following intraperitoneal versus intravenous injection at 0.5, 3 and 24 h of circulation time.

Methods

58 CD-1 mice were used. Animals were divided into ICH (N = 42), sham groups (N = 6) and naïve (N = 10). ICH animals received stereotactic injection of collagenase type VII into the right basal ganglia. Sham animals received only needle trauma. Evans Blue stain was injected 24 h after collagenase injection or needle trauma. The consistency of ICH produced was characterized by estimation of hematoma volume via hemoglobin assay and neurological evaluation.

Results

The produced hematoma and neurological deficits were well comparable between different experimental groups. There was no statistically significant difference in the results of the Evans Blue assay with regard to administration route.

Conclusions

The amount of Evans Blue stain accumulated in the brains of mice after ICH produced by collagenase injection was independent of the stain administration route.

Introduction

The blood–brain barrier (BBB) is a specialized vascular system consisting of endothelial cell tight junctions, basal lamina and glial processes (Rubin and Staddon, 1999). It separates circulating blood from cerebrospinal fluid in the central nervous system and has a low permeability to ionized water-soluble molecules with a molecular mass greater than 180 Da, unless the molecule can cross into the brain via specific active transporters (Kroll and Neuwelt, 1998). Disruption of the BBB following brain injury results in the development of vasogenic brain edema, a most life threatening event after such events as ICH (Xi et al., 2006). Since preservation of the BBB is a common goal among neuroprotective therapies, an objective method for evaluating blood brain barrier disruption is needed.

A common technique of evaluation involves the staining of plasma serum albumin, known as the Evans Blue assay. The major characteristic of Evans Blue stain is its ability to bind to serum albumin immediately after stain injection into the blood stream (Reeve, 1965). Since plasma albumin does not pass the BBB under normal physiologic conditions, spectrophotometric determination of Evans Blue stain accumulation is an easy and reliable way to estimate BBB permeability. In this study we compared different protocols of this assay and evaluated the difference in the Evans Blue stain accumulation in brain of mice after intraperitoneal versus intravenous injection of dry stain, at three different points of circulation time.

Section snippets

Animal groups and treatment methods

This study was conducted in accordance with the National Institutes of Health guidelines for the treatment of animals and was approved by the Animal Care and Use Committee at Loma Linda University. Male CD-1 mice (34–43 g, Charles River, MA, USA) were housed with a 12-h light/dark cycle with access to water and food ad libitum. A total of 58 mice were used. Animals were divided into two groups: a sham (needle-trauma only) group (6 mice) and an ICH group (42 mice). All animals received an

Results

No neurological deficit between ICH animals in different groups was observed.

Results of neurological testing are summarized in Fig. 1A–C. All animals that received collagenase injections demonstrated significant neurological deficit (p < 0.05 vs. sham). No statistically significant difference between experimental groups was observed.

Discussion

The aim of this study was to establish whether the administration route or circulation time would affect the amount of Evans Blue delivered across the blood–brain-barrier into the brain of mice after ICH.

Brain edema is most devastating life-threatening complication after ICH. Multiple forms of edema are present after ICH, but the main form is likely vasogenic (Xi et al., 2006, Adeoye et al., 2010) in origin. Several mechanisms have been proposed to be responsible for the development of

Conclusion

Evans Blue stain accumulation in the brains of mice after ICH is independent from administration route and circulation time.

Acknowledgment

We would like to thank Suzzanne Marcantonio (the Department of Anesthesiology, Loma Linda University) for excellent technical assistance.

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Comparison Evans Blue injection routes: Intravenous versus intraperitoneal, for measurement of blood–brain barrier in a mice hemorrhage model

Abstract

Aims

Methods

Results

Conclusions

Introduction

Section snippets

Animal groups and treatment methods

Results

Discussion

Conclusion

Acknowledgment

J. Biol. Chem.

Neurosci Lett

Neurochem Int

Exp Neurol

Lancet Neurol

Do current animal models of intracerebral hemorrhage mirror the human pathology?

Transl Stroke Res

Albumin treatment reduces neurological deficit and protects blood–brain barrier integrity after acute intracortical hematoma in the rat

Stroke

Use of a spectrophotometric hemoglobin assay to objectively quantify intracerebral hemorrhage in mice

Stroke

Blood–brain barrier permeability and monocyte infiltration in experimental allergic Encephalomyelitis: a quantitative MRI study

Brain

Rapid reversal of anticoagulation reduces hemorrhage volume in a mouse model of Warfarin-associated intracerebral hemorrhage

J Cereb Blood Flow Metab

Enlargement of spontaneous intracerebral hemorrhage incidence and time course

Stroke

Decreased brain edema after collagenase-induced intracerebral hemorrhage in mice lacking the inducible nitric oxide synthase gene

J Neurosurg

Altered blood–brain barrier permeability and its effect on the distribution of Evans Blue and sodium fluorescein in the rat brain applied by intracarotid injection

Physiol Res